1. Field of the Invention
The instant invention pertains to an electrophoretic technique for the separation of lipoproteins and to an electrophoretic gel for use therein.
2. Description of the Prior Art
Electrophoresis involves the placing of a sample substance, such as blood serum or urine, in a support medium across which is maintained a direct current electrical potential. Support media characteristically include paper, agar; agarose; cellulose acetate and polyacrylamide. The electrical potential causes the colloidal particles in the sample substance to migrate toward one or the other electrodes. The amount of migration is determined by the electrical charges on the particles in the sample substance and the magnitude of the imposed electrical potential. Particles with similar properties tend to group into defined areas and thus a determination can be made as to the amount of each class of substance present in the sample. A graph or analog curve of the relative concentrations of particles can provide information as to the relative proportions of each which are contained in the sample substance. These electrophoretograms provide useful information as to blood serum or urine composition which may be used by clinical pathologists or the like.
More particularly, electrophoretic techniques for the separation of lipoproteins and electrophoretic gels for use therein are well known to those skilled in the art. See Bibliography (1-19) In general, electrophoretic gels employed for separating lipoproteins are of the type comprising a matrix selected from the group consisting of a polysaccharide and derivatives thereof. Examples of polysaccharides include, but are not limited to, agar, agarose, and mixtures thereof. Polysaccharide derivatives include, but are not limited to, cellulose acetate. Albumin is often, but not always, incorporated into the gel. In addition, a buffer having a basic pH is also commonly present in these electrophoretic gels. Barbital buffer (pH 8.6) is reported to yield the most satisfactory results.
Due to the great variety of electrophoretic cells and variety of dimensions of gel supporters, the specific electrophoretic conditions vary considerably. In general, electrophoretic conditions are specific for the design of the gel supporters and for the dimension of the cells.
After electrophoretically separating the lipoproteins, the lipoproteins are immobilized in the gel. Typical immobilization methods include fixation and precipitation.
The precipitation technique involves localizing the lipoproteins via the use of a precipitation solution. Numerous precipitation solutions are known to those skilled in the art. If the precipitation technique is employed, the lipoproteins in these gels can then be evaluated by known techniques, e.g., densitometry and integration.
The fixation method involves localizing the lipoprotein via the use of a fixative solution. In this step, one can use any one of the numerous, reported fixative solutions. The gels are then subjected to a drying step. This drying step dehydrates the gel. The dehydrated gel is customarily referred to as a "film".
The next step in this procedure entails the visualization of the fixed lipoproteins in the film. Staining techniques are a commonly employed visualization methodology.
After the staining operation, destaining is the next step which is performed on the film. Various solutions and solvents capable of use in destaining methodologies have been reported.
The films are again dried via one of numerous known techniques.
The stained lipoproteins in the dried films can then be evaluated by known techniques, e.g., densitometry and integration.
Densitometers are well known devices which scan a sample and provide an output signal or graphical display indicative of the optical density, transmittance, absorption or the like of the scanned sample.
One well known use of the densitometer is to scan a sample of blood which has been prepared by the electrophoresis process. As noted above, electrophoresis of blood samples separates various proteins in the blood from each other. Each of these separated proteins exhibit light absorption characteristics based upon the density of each protein and the light absorption patterns are graphically displayed by the densitometer to indicate the presence and quantity of each of these proteins via a series of peaks and valleys as shown, for example, in FIG. 1.
There has been much discussion in the literature concerning the nature of the lipid bands migrating in the alpha electrophoretic zone (6-9, 11, 12, 14-16). While some authors feel that there are two lipoprotein bands migrating in the alpha zone (12), designated as alpha lipoprotein and pre-alpha lipoprotein, the majority of authors feel the faster moving alpha band does not contain a lipoprotein but is instead serum albumin containing bound free fatty acids (11, 12, 16). With this latter position in mind, one problem in the use of known prior art gel formulations for the electrophoretic separation of lipoproteins is that the alpha lipoprotein band 106, as shown in FIG. 1, does not separate from the free fatty acid albumin band 108. As a result, when the electrophoretic gel is stained for detection of lipoproteins by lipid dyes, the free fatty acid albumin band 108, which is also stained by lipid dyes, interferes with the evaluation of the alpha lipoprotein band 106.
Accordingly, it would be very desirable to have an electrophoretic gel for use in the separation of lipoprotein which is capable of separating the alpha lipoprotein band 106 from the free fatty acid albumin band 108.